An innovative lyophilized dry powder formulation consisting of urea-crosslinked hyaluronic acid (HA-CL) and sodium ascorbyl phosphate (SAP) – LYO HA-CL – SAP- was prepared and characterized in vitro for physico-chemical and biological properties. The aim was to understand if LYO HA-CL – SAP could be used as adjuvant treatment for nasal inflammatory diseases. LYO HA-CL – SAP was suitable for nasal delivery and showed to be not toxic on human nasal septum carcinoma-derived cells (RPMI 2650 cells) at the investigated concentrations. It displayed porous, polygonal particles with unimodal, narrow size distribution, mean geometric diameter of 328.3 ± 27.5 µm, that is appropriate for nasal deposition with no respirable fraction and 88.7% of particles with aerodynamic diameter >14.1 µm. Additionally, the formulation showed wound healing ability on RPMI 2650 cells, and reduced interleukin-8 (IL-8) level in primary nasal epithelial cells pre-induced with lipopolysaccharide (LPS). Transport study across RPMI 2650 cells showed that HA-CL could act not only as carrier for SAP and active ingredient itself, but potentially also as mucoadhesive agent. In conclusion, these results suggest that HA-CL and SAP had anti-inflammatory activity and acted in combination to accelerate wound healing. Therefore, LYO HA-CL – SAP could be a potential adjuvant in nasal anti-inflammatory formulations.

This study investigated how varying the dosing cup size of a novel reservoir dry powder inhaler (DPI) affects the detachment of a micronized active pharmaceutical ingredient from larger carrier particles, and the aerosol performance of a DPI carrier formulation. Three different-sized dosing cups were designed: 3D printed with cup volumes of 16.26 mm3, 55.99 mm3, and 133.04 mm3, and tested with five different carrier type formulations with beclomethasone dipropionate (BDP) concentrations between 1% and 30% (w/w). The morphology of the BDP attached to the carrier was investigated using scanning electron microscopy and the aerosol performance using the Next Generation Impactor. Increasing the volume of the dosing cup led to a reduction of BDP deposition in the Next Generation Impactor preseparator, and an increase in BDP detachment from the carrier was observed, leading to increased aerosol performance. The decreased amount of BDP attached to carrier after aerosolization was attributed to the increased dosing cup void volume. This may enable greater particle-particle and particle-wall collisions, with greater BDP detachment from the carrier and deagglomeration of smaller agglomerates. The dosing cup volume was observed to have significant influence on particle dispersion and the overall aerosol performance of a DPI.

The use of cell-penetrating peptides (CPPs) in combination with nanoparticles (NPs) shows great potential for intracellular delivery of DNA. Currently, its application is limited due to the potential toxicity and unknown long-term side effects. In this study NPs prepared using a biodegradable polymer, poly(lactic–co–glycolic acid (PLGA) in association with a CPP, was assessed on two lung epithelial cell lines (adenocarcinomic human alveolar basal epithelial cells (A549) and normal bronchial epithelial cells (Beas-2B cells)). Addition of CPP was essential for intracellular internalization. No effects were observed on the mitochondrial activity and membrane integrity. Cells exposed to the NPs–DNA–CPP showed low inflammatory response, low levels of apoptosis and no activation of caspase-3. Increase in necrotic cells (between 10%–15%) after 24 h of incubation and increase in autophagy, induced by NPs–DNA–CPP, are likely to be related to the lysosomal escape mechanism. Although oxidative stress is one of the main toxic mechanisms of NPs, NPs–DNA–CPP showed decreased reactive oxygen species (ROS) production on Beas-2B cells, with potential antioxidant effect of CPP and no effect on A549 cells. This NP system appears to be safe for intracellular delivery of plasmid DNA to the lung epithelial cells. Further investigations should be conducted in other lung-related systems to better understand its potential effects on the lungs.

Fluticasone propionate uptake in the presence of a proprietary cell-penetrating peptide (human stimulus factor, [HSF]) based on the N-terminal domain of lactoferrin was studied, alone and in combination with salmeterol, using an air interface Calu-3 epithelial model. The HSF enhanced uptake and transport of fluticasone propionate across the epithelial barrier when alone and in presence of salmeterol. This was attributed to transcellular-mediated uptake. This HSF is a promising peptide for delivery of therapeutics where enhanced epithelial penetrating is required.

PURPOSE:

In this study, a cell penetrating peptide was used as an uptake enhancer for pDNA delivery to the lungs.

METHODS:

Polyplexes were prepared between pDNA and CPP. Intracellular delivery of pDNA was assessed in both alveolar (A549) and bronchial (Calu-3) epithelial cells. Aerosol delivery was investigated using a mesh nebulizer.

RESULTS:

Efficient intracellular delivery of pDNA occurs in both A549 and Calu-3 cells when delivered as polyplexes. Protection against nucleases and endosomal escape mechanism occurs when pDNA is formulated within the polyplexes. For aerosol delivery, 1% (w/v) mannitol was able to protect naked DNA structure during nebulization with a significant increase in fine particle fraction (particles <5 μm). The structure of polyplexes when delivered via a mesh nebulizer using 1% (w/v) mannitol could partially withstand the shear forces involved in aerosolization. Although some loss in functionality occurred after nebulization, membrane-associated fluorescence was observed in A549 cells. In Calu-3 cells mucus entrapment was a limiting factor for polyplex delivery.

CONCLUSIONS:

The presence of CPP is essential for efficient intracellular delivery of pDNA. The polyplexes can be delivered to lung epithelial cells using mesh nebulizer. The use of different excipients is essential for further optimization of these delivery systems.

The purpose of this study was to present a novel and simple drug deposition method to evaluate drug transport of aerosol microparticles across airway epithelial cells. Microparticles containing ciprofloxacin HCl (Cip) and doxycycline (Dox), alone or in a 50:50% w/w ratio, were spray dried and suspended using 2H, 3H-perfluoropentane, model propellant. The suspension was then used to assess deposition, and transport of these drug microparticles across sub-bronchial epithelial Calu-3 cells was also studied. In comparison with other methods of depositing microparticles, this proposed method, using drug suspended in HPFP, provides control over the amount of drugs applied on the surface of the cells. Therefore, cell permeability studies could be conducted with considerably smaller and more reproducible doses, without the physicochemical characteristics of the drugs being compromised or the use of modified pharmacopeia impactors. The suspension of microparticles in HPFP as presented in this study has provided a non-toxic, simple, and reproducible novel method to deliver and study the permeability of specific quantity of drugs across respiratory epithelial cells in vitro.

Airway stents are commonly used in the management of patients suffering from central airway obstruction (CAO). CAO may occur directly from airway strictures, obstructing airway cancers, airway fistulas or tracheobronchomalacia, resulting from the weakening and dynamic collapse of the airway wall. Current airway stents are constructed from biocompatible medical-grade silicone or from a nickel–titanium (nitinol) alloy with fixed geometry. The stents are inserted via the mouth during a bronchoscopic procedure. Existing stents have many shortcomings including the development of obstructing granulation tissue in the weeks and months following placement, mucous build up within the stent, and cough. Furthermore, airway stents are expensive and, if improperly sized for a given airway, may be easily dislodged (stent migration). Currently, in Australia, it is estimated that approximately 12,000 patients will develop CAO annually, many of whom will require airway stenting intervention. Of all stenting procedures, the rate of failure is currently reported to be at 22%. With a growing incidence of lung cancer prevalence globally, the need for updating airway stent technology is now greater than ever and personalizing stents using 3D-printing technology may offer the best chance of addressing many of the current limitations in stent design. This review article will assess what represents the gold standard in stent manufacture with regards to treatment of tracheobronchial CAO, the challenges of current airway stents, and outlines the necessity and challenges of incorporating 3D-printing technology into personalizing airway stents today.

Lung cancer is a highly invasive and prevalent disease with ineffective first-line treatment and remains the leading cause of cancer death in men and women. Despite the improvements in diagnosis and therapy, the prognosis and outcome of lung cancer patients is still poor. This could be associated with the lack of effective first-line oncology drugs, formation of resistant tumors and non-optimal administration route. Therefore, the repurposing of existing drugs currently used for different indications and the introduction of a different method of drug administration could be investigated as an alternative to improve lung cancer therapy. This review describes the rationale and development of repositioning of drugs for lung cancer treatment with emphasis on inhalation. The review includes the current progress of repurposing non-cancer drugs, as well as current chemotherapeutics for lung malignancies via inhalation. Several potential non-cancer drugs such as statins, itraconazole and clarithromycin, that have demonstrated preclinical anti-cancer activity, are also presented. Furthermore, the potential challenges and limitations that might hamper the clinical translation of repurposed oncology drugs are described.

This in vitro study evaluated, for the first time, the safety and the biological activity of a novel urea-crosslinked hyaluronic acid component and sodium ascorbyl phosphate (HA-CL – SAP), singularly and/or in combination, intended for the treatment of inflammatory lung diseases. The aim was to understand if the combination HA-CL – SAP had an enhanced activity with respect to the combination native hyaluronic acid (HA) – SAP and the single SAP, HA and HA-CL components. Sample solutions displayed pH, osmolality and viscosity values suitable for lung delivery and showed to be not toxic on epithelial Calu-3 cells at the concentrations used in this study. The HA-CL – SAP displayed the most significant reduction in interleukin-6 (IL-6) and reactive oxygen species (ROS) levels, due to the combined action of HA-CL and SAP. Moreover, this combination showed improved cellular healing (wound closure) with respect to HA – SAP, SAP and HA, although at a lower rate than HA-CL alone. These preliminary results showed that the combination HA-CL - SAP could be suitable to reduce inflammation and oxidative stress in lung disorders like acute respiratory distress syndrome, asthma, emphysema and chronic obstructive pulmonary disease, where inflammation is prominent.

The pulmonary route of administration has been commonly used for local lung conditions such as asthma and chronic obstructive pulmonary disease (COPD). Recently, with the advent of new technologies available for both formulation and device design, molecules usually delivered at high doses, such as antibiotics and insulin to treat cystic fibrosis (CF) and diabetes, respectively, can now be delivered by inhalation as a dry powder. These molecules are generally delivered in milligrams instead of traditional microgram quantities. High dose delivery is most commonly achieved via dry powder inhalers (DPIs), breath activated devices designed with a formulated powder containing micronized drug with aerodynamic diameters between 1 and 5 µm. The powder formulation may also contain other excipients and/or carrier particles to improve the flowability and aerosol dispersion of the powder. A drawback with high doses is that the formulation contains a great number of fine particles, leading to a greater degree of cohesive forces, producing strongly bound agglomerates. With greater cohesive forces holding fine particles together, higher dispersion forces are needed for efficient de-agglomeration and aerosolisation. This requirement of greater dispersion forces has led to different dry powder formulations and vastly different inhaler designs. The purpose of this review is to evaluate the different formulation types, various DPI devices currently available, and how these affect the aerosolisation process and delivery of high dosed inhalable dry powder formulations to the lungs.

Background: Both obesity and high dietary fat intake activate the nucleotide oligomerization domain–like receptor protein 3 (NLRP3) inflammasome.

Objective: We aimed to examine NLRP3 inflammasome activity in the airways of obese asthmatic patients after macronutrient overload and in immune cells challenged by inflammasome triggers.

Methods: Study 1 was a cross-sectional observational study of nonobese (n = 51) and obese (n = 76) asthmatic adults. Study 2 was a randomized, crossover, acute feeding study in 23 asthmatic adults (n = 12 nonobese and n = 11 obese subjects). Subjects consumed 3 isocaloric meals on 3 separate occasions (ie, saturated fatty acid, n-6 polyunsaturated fatty acid, and carbohydrate) and were assessed at 0 and 4 hours. For Studies 1 and 2, airway inflammation was measured based on sputum differential cell counts, IL-1β protein levels (ELISA), and sputum cell gene expression (Nanostring nCounter). In Study 3 peripheral blood neutrophils and monocytes were isolated by using Ficoll density gradient and magnetic bead separation and incubated with or without palmitic acid, LPS, or TNF-α for 24 hours, and IL-1β release was measured (ELISA).

Results: In Study 1 NLRP3 and nucleotide oligomerization domain 1 (NOD1) gene expression was upregulated, and sputum IL-1β protein levels were greater in obese versus nonobese asthmatic patients. In Study 2 the saturated fatty acid meal led to increases in sputum neutrophil percentages and sputum cell gene expression of Toll-like receptor 4 (TLR4) and NLRP3 at 4 hours in nonobese asthmatic patients. In Study 3 neutrophils and monocytes released IL-1β when challenged with a combination of palmitic acid and LPS or TNF-α.

Conclusion: The NLRP3 inflammasome is a potential therapeutic target in asthmatic patients. Behavioral interventions that reduce fatty acid exposure, such as weight loss and dietary saturated fat restriction, warrant further exploration.

Despite many therapeutic advancements over the past decade, the continued rise in chronic inflammatory lung diseases incidence has driven the need to identify and develop new therapeutic strategies, with superior efficacy to treat these diseases. Statins are one class of drug that could potentially be repurposed as an alternative treatment for chronic lung diseases. They are currently used to treat hypercholesterolemia by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, that catalyses the rate limiting step in the mevalonate biosynthesis pathway, a key intermediate in cholesterol metabolism. Recent research has identified statins to have other protective pleiotropic properties including anti-inflammatory, anti-oxidant, muco-inhibitory effects that may be beneficial for the treatment of chronic inflammatory lung diseases. However, clinical studies have yielded conflicting results. This review will summarise some of the current evidences for statins pleiotropic effects that could be applied for the treatment of chronic inflammatory lung diseases, their mechanisms of actions, and the potential to repurpose statins as an inhaled therapy, including a detailed discussion on their different physical-chemical properties and how these characteristics could ultimately affect treatment efficacies. The repurposing of statins from conventional anti-cholesterol oral therapy to inhaled anti-inflammatory formulation is promising, as it provides direct delivery to the airways, reduced risk of side effects, increased bioavailability and tailored physical-chemical properties for enhanced efficacy.

Purpose: This study was performed to investigate how increasing the active pharmaceutical ingredient (API) content within a formulation affects the dispersion of particles and the aerosol performance efficiency of a carrier based dry powder inhalable (DPI) formulation, using a custom dry powder inhaler (DPI) development rig.

Methods: Five formulations with varying concentrations of API beclomethasone dipropionate (BDP) between 1% and 30% (w/w) were formulated as a multi-component carrier system containing coarse lactose and fine lactose with magnesium stearate. The morphology of the formulation and each component were investigated using scanning electron micrographs while the particle size was measured by laser diffraction. The aerosol performance, in terms of aerodynamic diameter, was assessed using the British pharmacopeia Apparatus E cascade impactor (Next generation impactor). Chemical analysis of the API was observed by high performance liquid chromatography (HPLC).

Results: Increasing the concentration of BDP in the blend resulted in increasing numbers and size of individual agglomerates and densely packed BDP multi-layers on the surface of the lactose carrier. BDP present within the multi-layer did not disperse as individual primary particles but as dense agglomerates, which led to a decrease in aerosol performance and increased percentage of BDP deposition within the Apparatus E induction port and pre-separator.

Conclusion: As the BDP concentration in the blends increases, aerosol performance of the formulation decreases, in an inversely proportional manner. Concurrently, the percentage of API deposition in the induction port and pre-separator could also be linked to the amount of micronized particles (BDP and Micronized composite carrier) present in the formulation. The effect of such dose increase on the behaviour of aerosol dispersion was investigated to gain greater insight in the development and optimisation of higher dosed carrier-based formulations.

A limitation in the systemic uptake of many inhalable drugs is the restricted permeation through the pulmonary epithelial layer barrier. One strategy to bypass the epithelial layer when delivering non-permeable drugs is to alter the paracellular transport, allowing the uptake of drugs into the systemic circulation. In this study, the potential of sodium decanoate (Na dec), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) as absorption enhancers has been investigated to increase pulmonary paracellular permeability by modulating epithelial cells' tight junctions. By incorporating Na dec, DHA and EPA, separately, into a nebulising formulation, the aim was to enhance the absorption of a fluorescent marker (flu-Na, used as model drug) across pulmonary epithelial cells (Calu-3). Results indicate that the aerosol performance of all the nebulizing formulations containing absorption enhancers was significantly better than control. Furthermore, the in vitro cell assays demonstrated a significant increase in paracellular transport of the fluorescent marker with Na dec and DHA formulations. This finding supports the potential use ofDHA and Na dec to enhance epithelial transport of poorly permeable drugs delivered via inhalation.

Purpose: The failure of chronic therapy with antibiotics to clear persistent respiratory infection is the key morbidity and mortality factor for patients with chronic lung diseases, primarily due to the presence of biofilm in the lungs. It is hypothesised that carbon sources, such as mannitol, could stimulate the metabolic activity of persister cells within biofilms and restore their susceptibility to antibiotics. The aims of the current study are to: (1) establish a representative in vitro model of Pseudomonas aeruginosa biofilm lung infection, and (2) investigate the effects of nebulised mannitol on antibiotic efficacy, focusing on ciprofloxacin, in the eradication of biofilm.

Method: Air interface biofilm was cultured onto Snapwell inserts incorporated into a modified pharmacopeia deposition apparatus, the Anderson Cascade Impactor (ACI). Three different formulations including mannitol only, ciprofloxacin only and combined ciprofloxacin and mannitol were nebulised onto the P. aeruginosa biofilm using the modified ACI. Antibacterial effectiveness was evaluated using colony-forming units counts, biofilm penetration and scanning electron microscopy.

Results: Nebulised mannitol promotes the dispersion of bacteria from the biofilm and demonstrated a synergistic enhancement of the antibacterial efficacy of ciprofloxacin compared to delivery of antibiotic alone.

Conclusions: The combination of ciprofloxacin and mannitol may provide an important new strategy to improve antibiotic therapy for the treatment of chronic lung infections. Furthermore, the development of a representative lung model of bacterial biofilm could potentially be used as a platform for future new antimicrobial pre-clinical screening.

Oil-in-water emulsion droplets, containing an elastic endoskeleton that holds the droplets in various non-spherical shapes, are formed by crystallizing a portion of the oil phase into a network of wax crystals. Such structures have recently been found to provide enhanced active ingredient delivery and shape-changing responsiveness, but robust methods of producing such droplets are needed that enable control of droplet size and shape. A continuous microfluidic flow is used here to produce endoskeleton droplets whose size is controlled by fluid flow rate and whose shape is varied between spheres, ellipsoids, and rods by control of exit temperature. A wide range of anisotropic shapes is produced using a single flow channel geometry by allowing the endoskeleton droplet to relax its deformation by varying degrees in response to fluid interfacial tension. Flexible production of shaped endoskeleton droplets will expand their application in enhanced delivery, deposition testing, and additive manufacturing processes.

Drug delivery by inhalation offers several advantages compared to other dosage forms, including rapid clinical onset, high bioavailability, and minimal systemic side effects. Drug delivery to the lung can be achieved as liquid suspensions or solutions in nebulizers and pressurized metered-dose inhalers (pMDI), or as dry powders in dry powder inhalers (DPIs). Compared to other delivery systems, DPIs are, in many cases, considered the most convenient as they are breath actuated and do not require the use of propellants. Currently, the delivery of low drug doses for the treatment of lung conditions such as asthma and chronic obstructive pulmonary disease are well established, with numerous commercial products available on the market. The delivery of low doses can be achieved from either standard carrier- or aggregate-based formulations, which are unsuitable in the delivery of high doses due to particle segregation associated with carrier active site saturation and the cohesiveness of micronized aggregates which have poor flow and de-agglomeration properties. High-dose delivery is required for the treatment of lung infection (i.e. antibiotics) and in the emerging application of drug delivery for the management of systemic conditions (i.e. diabetes). Therefore, there is a demand for new methods for production of high-dose dry powder formulations. This paper presents a review of co-mill processing, for the production of high-efficiency inhalation therapies, including the jet mill, mechanofusion, or ball mill methodologies. We investigate the different techniques, additives, and drugs studied, and impact on performance in DPI systems.

Introduction: Cystic fibrosis (CF) is a congenital life-limiting, orphan disease affecting 1/2500 – 1/3000 people worldwide with the greatest prevalence in Europe, North America and Australia. The primary reason underpinning the cause of morbidity and mortality of CF patients is associated with recurrent pulmonary inflammation and infection that leads to chronic, progressive lung deterioration and ultimately death of CF patients.

Areas covered: This review aims to explore the potential role for inhaled anti-inflammatory drugs as a more successful treatment option for CF, in comparison with current oral delivery. Specifically, the focus is on ibuprofen, the only nonsteroidal anti-inflammatory drug approved for chronic use in CF. The need for inhalation therapy has also been highlighted with an insight on the reasons and challenges associated with developing an inhalation therapy of nonsteroidal anti-inflammatory drugs (NSAIDs).

Expert opinion: There is a fundamental need to direct research towards development of anti-inflammatory drugs to control inflammation rather than just targeting infection. Development of an inhalable preparation of ibuprofen alone or in combination with an antibiotic holds the potential to be the most effective treatment option among the existing array of therapies available for CF.

Aim: Current inhaled treatments are not adequate to treat lung diseases. In this study, a promising nanotechnology has been developed to deliver a potential anti- inflammatory and muco-inhibitory compound, simvastatin, for treatment of inflammatory lung diseases via inhalation.

Material & methods: Simvastatin nanoparticles (SV-NPs) encapsulated with poly (Lactic-Co-Glycolic) acid were fabricated using the solvent and anti-solvernt precipitation method.

Results: SV-NPs were found to be stable up to 9 months at 4°C in a freeze-dried form prior to reconsititution. The amount of mucus produced was significantly reduced after SV-NPs treatment on inflamed epithelial cell model and were effective in suppressing the pro-inflammatory markers expression.

Conclusion: This study suggests that SV-NPs nebulisation could potentially be used for the treatment of chronic pulmonary diseases.

In this study, the development and validation of a novel modified version of the medium-sized Virginia Commonwealth University (VCU) mouth-throat (MT) and tracheal-bronchial (TB) realistic upper airway model, intended to evaluate total airway deposition and drug permeation behaviour for orally inhaled products (OIPs) in vitro, is presented. The VCU MT-TB model was modified to accommodate two Snapwell® permeable membrane inserts above the first TB airway bifurcation region to primarily collect deposited nebulised ciprofloxacinhydrochloride (CIP-HCL) droplets as a model drug aerosol system. Permeation characteristics were assessed by adapting the established Transwell® ‘fluid-capacity-limited’ dissolution test system. Firstly, the novel modified TB airway model resulted in significantly higher TB aerosol deposition compared with the original TB model. Secondly, the adapted Transwell permeation test system demonstrated reproducible and discriminatory permeation profiles for already-dissolved and nebulised CIP-HCL drug through a range of permeable membranes. Fundamentally, the rate and extent of CIP-HCL permeation depended on the permeable membrane material used, presence of a stirrer in the receptor compartment and, most importantly, the aerosol particle collection method. This novel hybrid in vitro approach, which incorporates a modified realistic airway model coupled with an adapted Transwell system holds great potential to evaluate other post-deposition characteristics, such as particle dissolution and cellular uptake of OIPs.